The Role of the Steroid Nuclear Receptor Genes in Breast Cancer

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Abstract

Breast cancer is a great source of morbidity and mortality in the developed world, being the most common cause of cancer death in Australian women and affecting roughly 1 in 10 women. The development and progression of cancers is a multi-stage process, involving numerous perturbations to normal cellular functions, especially to those genes which control cellular growth, cellular differentiation and DNA repair. Over time, these alterations combine to change normal cells into cancerous ones that typically no longer respond to normal control stimuli and grow with great rapidity. The nuclear receptors are a family of proteins which accept incoming signals from various molecules, and then alter gene expression or affect cell behavior directly. The steroid nuclear receptors receive transducer signals from hormones such as estrogen and testosterone and are intimately involved in affecting cellular growth and differentiation. Stimulation of the steroid receptors have been used as successful treatment avenues, so how these genes behave in cancer is of great interest. Accordingly, this study has examined the expression of various nuclear receptors and some related genes in a number of tissue samples derived from breast tumours and from surrounding areas, and examined how various pathological parameters affect their expression. Tissue samples were first microdissected to separate tumour tissue from the surrounding stroma and then subjected to RNA extraction procedures to allow gene expression to be quantitated. RNA was then converted into cDNA by reverse transcription and the genes of interest amplified and quantitated using semiquantitative polymerase chain reaction. Expression data was then normalized by expressing it as a ratio of the gene of interest to the 18S ribosomal gene and differences in expression analysed by analysis of variance (ANOVA) testing. The first portion of the study dealt with the progesterone and glucocorticoid receptors in tumour tissue. Progesterone is an anti-mitogenic signal for breast tissue and the stimulation of the receptor is a useful part of combined hormonal therapy for breast cancer. The glucocorticoid receptor plays a similar role to the progesterone receptor, slowing breast tissue growth and promoting apoptosis. The complete tissue population of 25 samples from control, grade 1, grade 2 and grade 3 tumours underwent PCR for the glucocorticoid and progesterone receptor genes and was normalized using 18S. Analysis of the expression data by ANOVA showed that the progesterone and glucocorticoid receptors were more highly expressed in grade 3 tumour tissue (p=0.023 and p=O.00033, for the progesterone and glucocorticoid receptors, respectively). Most advanced tumours cease being responsive to growth repressing hormones, so these results indicated that the control of the expression of these receptors in tumour tissue was more complicated than might have been expected. The increase in expression observed may be the result of intact growth preventing feedback mechanisms which have malfunctioned in some manner. There are several mechanisms the tumours may be using to escape an increase in progesterone or glucocorticoid sensitivity. The mRNA detected may simply not be being translated into completed proteins or be being spliced into isoforms of the receptors that favour tissue growth. The second portion of the study dealt with the estrogen receptors alpha and beta in tumour tissue. The estrogen receptors control estrogen signaling and are highly important in breast cancer, since estrogen is the primary growth inducing hormone for breast tissue. For the estrogen receptors alpha and beta, the complete tissue population of 25 samples from control, grade 1, grade 2 and grade 3 tumours underwent PCR and were normalized using 18S. ANOVA analysis found that the expression of the estrogen receptors did not change significantly in any grade of cancer (p= 0.057 and p=O.7'38, for ESR&alpha; and ESR&beta;, respectively) nor in tissues that are negative for the estrogen receptor alpha protein, a poor prognostic factor in breast cancer (p= 0.794 and p=O.7l6, for ESR&alpha; and ESR&beta;, respectively). These results indicated that the loss of estrogen receptor in advanced cancers is not controlled at the level of mRNA. The mRNA observed in this study may be being spliced into alternate and possibly inactive isoforms, or may be being degraded post transcriptionally, preventing estrogen stimulation in these tumours. This could prove an excellent area for further study, since if the mechanism that prevents or distorts ESR mRNA translation can be discovered, it would allow manipulation of one of the most important treatment avenues for breast cancer. The third portion of the study dealt with the expression of the androgen receptor in tumour tissue. Androgens are a strong anti-growth signal in breast tissue and despite the side effects that androgens have on women, they have been used to treat breast cancer with some success. The complete tissue population of 25 samples from control, grade 1, grade 2 and grade 3 tumours underwent PCR for the androgen receptor gene and normalized using 18S. The results obtained for androgen receptor expression in tumour tissue showed that androgen receptor expression was significantly elevated in grade 2 and grade 3 tumour tissue, as well as in ESR&alpha; negative tumours (p= 0.014 and p=O.O25, respectively). An increase in expression in late stage tumours would seem to be unusual for an anti-mitogenic receptor, however many advanced breast tumours have been found to be receptive to androgen stimulation, even when they no longer respond to other hormones. The increased expression of AR may be a normal response to cellular over-growth, or it may be a mechanism by which the tumour prevents stimulation by other growth retarding hormones, by sequestering all available receptor co-enzymes with a receptor that is unlikely to be stimulated. The fourth portion of the study examined the expression of the estrogen alpha, estrogen beta, progesterone, glucocorticoid and androgen nuclear receptors in stromal tissue derived from the tumours studied in previous chapters. Tumours have been observed in other studies to manipulate the activities of the cells that surround them through the release of cofactors and vice versa. These cofactors include the steroid hormones, among others, and hence the study of how the tumour and stroma interacts is a valuable extension to the results obtained in the previous sections. PCR was performed for all nuclear receptors, except for the estrogen receptor alpha, in the complete tissue population of 25 samples of tissue derived from the stroma of the grade 1, grade 2 and grade 3 tumours used in the previous studies as well as the control tissues. Due to difficulties in PCR optimization for estrogen receptor alpha, only three stromal samples from each grade and four controls were able to produce results, for a total population of 13 samples. Of all the receptors tested, only the progesterone and glucocorticoid receptors displayed significant changes in expression in stromal tissue, with PgR having significantly lower expression in all stromal samples compared to control, while GR was more highly expressed in stroma derived from high grade tumours (p= 5.908x107 and p=2.761x105, for PgR and GR, respectively). UR expression was also increased in stroma derived from ESR&alpha; negative tumours (p=5.85x105). These alterations reflect the kind of stimulation a tumour is likely to apply to the surrounding stroma, using progesterone to stimulate the cells into differentiating to provide a more suitable environment, hence the loss in PgR expression. The increase in GR expression may be the result of the high level of growth stimulating factors that tumours produce, priming the local cells to be more sensitive to growth suppressors, a situation that is also mirrored in results previously obtained for the tumour tissue. The fifth part of the study concerned the expression of the nuclear receptor coactivator 1 and nuclear receptor co-activator 3 genes. These proteins are required for the activation and function of the nuclear receptors and both have been implicated in cancer development, being found to be over-expressed in several tissues and cell lines. As integral parts of the nuclear receptor pathway, their level of expression is important for determining how effective any nuclear receptors present will be when stimulated. The complete tissue population of 25 samples from control, grade 1, grade 2 and grade 3 tumours underwent PCR for both nuclear receptor co-activator genes and normalized using 18S. The result of ANOVA analysis on the NCoA data showed that NCoA3 expression remained unaltered in all grades of cancer and stroma and in both ESR&alpha; positive and negative tissue. NCoA1 however, was significantly upregulated in grade 3 tumours as compared to grade 1 tumours and also in ESR&alpha; negative tumours. This increase in expression would seem to indicate that these tissues would be more capable of acting on any received hormonal stimulation. That this increase in expression occurs in more advanced cancers could be evidence that the nuclear receptor expression observed in prior sections is resulting in NR splice
variants that favour, rather than repress, growth, as advanced cancers usually do not respond normally to hormonal stimulation. The final part of the study investigated the possibility of correlations between the expression of the nuclear receptors, between the nuclear receptor co-activators and between all of the tested genes and other pathological parameters, including tumour size, metastasis, site of tumour, carcinoma in situ invasiveness, age of patient and the presence of calcification. The data generated in the prior studies was analyzed using ANOVA for categorical data and correlation analysis for numerical data. The ANOVA and correlation analysis revealed a number of interactions between these factors, which provide additional information on the relationships between the tested genes. Expression of the progesterone receptor was found to be correlated with the expression of GR, AR and NCoA1 (p= 0.022, p=O.OO3, and p=O.0i9, respectively). Likewise the expression of GR and AR were found to be correlated (p=O.O29). Additionally, AR was found to be associated with tumour size (p=O.O36) while GR was found to be associated with both tumour size and metastasis (p= 0.006 and p=7.6x106, respectively). ESR&alpha; and ESR&beta; expression were found to be negatively correlated (p=O.O44M), as were patient age and the amount of ductal carcinoma in situ invasion. Given the results of previous analyses, it is not surprising that PgR, GR, AR and NCoA1 expression are related, and the negative correlations between ESR&alpha; and ESR&beta; expression, as well as between age and ductal carcinoma in situ invasion have been documented in other studies. Hence, these results provide reinforcement for previous observations, as well as providing new information, particularly on AR and GR.